Procedure,

1. Induced breeding

2. 1. Basis of Induced breeding
2. Induced reproduction
3. Historical background
4. The fish Pituitary
5. Hormones of pituitary related with development of gonads
and spawning
6. Induced breeding in carps by hypophysation
a. Brood stock management
b. Collection of pituitary gland
c. Preservation and storage of gland
d. Prep and preservation of extract
e. Determination of pituitary dosage
f. Ponds for induced breeding and breeding happas
g. Injections of pituitary extract to breeders
h. Spawning and fertilization
i. Stripping or artificial insemination

3. I) Basis for induced breeding
• Reproduction in fish-regulated by environmental
stimuli.
Environmental
stimuli
Brain hypothalamus
Pituitary gland
Gonadotropic
hormones
Sex steroidal
hormones
By sensory receptors
stimulates
Releasing hormones
Gonads
Regulation of secondary
sexual chtrs, nuptial
coloration, breeding
behavior
I) Provision of appropriate
environmental stimuli
II) Administration of
hormones for maturation and
release of gonads

4. II) Induced reproduction
• Under culture conditions, the required environmental
factors may not be available or persist for sufficient
length of time for spontaneous maturation to occur.
• This led to Induced reproduction or Hypophysation
technique, where pituitary homogenates are injected
into fish.
• This stimulates gonadotropin surge, by-passing
environmental variables.
• Inducement of spawning in fishes that do not ordinarily
breed under conditions of confinement.

5. III) Historical background
• In India, first attempt to induce spawn of carps by
administering mammalian pituitary extract by
made by Hamid Khan (1938) but without success.
• Chaudhuri and Alikunhi (1957) succeeded in
making Labeo rohita, C. mrigala, C. reba, L. bata
and Puntius sarana to breed by injecting them
with fish pituitary extract.
• Chaudhuri (1959) successfully carried out cross
breeding of 12 different sps by artificial
insemination.

6. III) Fish pituitary
• Pituitary gland or hypophysis-situated on ventral side of brain in
concavity of cranium called sella turcicia and covered by duramater.
Pituitary has 2 parts:
• Neurohypophysis (nervous part)
• Adenophypophysis (glandular region)
Pro-adenohypophysis (ant lobe, ant glandular region)
meso-adenohypophysis (middle lobe, middle glandular region)
Meta-adenohypophysis (posterior glandular region)

7. IV) Hormones of pituitary related with development
of gonads and spawning
Adenohypophyseal
hormones:
• GH
• Somatotropic hormone
• TSH
• GTH
• ACTH
• Prolactin
• Melanocyte stimulating
hormone
Neurohypophyseal
hormones:
• Oxytocin
• Vassopressin
• ADH

8. • GTH-play imp role in regulating development of
gonads and spawning.
• Gonads release: Androgen, estrogen and
progesterone.
• Further environmental factors like temp, rain
influence the gonads and other endocrine system

9. VI) Induced breeding in carps by hypophysation
1. Brood stock Management
• The brood pond
• Characteristic of ripe brood fish and their
selection for stocking
• Stocking rate
• Caring the brooders
• Manuring and maintenance of water quality in
brood-stocking ponds
• Supplementary feeding
• Rate of feeding

10. 2. Collection of pituitary gland
3. Preservation and storage of gland
4.Preparation and preservation of pituitary extract
5. Determination of Pituitary dosage
6.Ponds for induced breeding and breeding
Happas
7. Injections of pituitary extract to the breeders
8.Spawning and fertilization
9.Stripping or artificial insemination

11. The primary requirement of carp breeding is an
adequate stock of good breeders. The excellent brood
fish culture is the foremost essential material basis and
key factor for successful artificial propagation.
Brood stock: Group of mature individuals used in
aquaculture for breeding purposes
a. The brood Pond
Ponds of moderate size (0.5 to 1 ha) & depth (1.5-
2.0 m),
preferably rectangular, flat bottom.
Moderate water temp (27-32°C).
Exposure of 6-8 hrs light in a day at least for 2-3
months.
Inductive for
rapid gonadal
growth of carps

12. b. Characteristics of ripe brood fish and their
selection for stocking
• Smooth pectoral fin.
•Abdomen is soft and bulging.
•Pinkish vent.
•The vent of fully ripe female
is slightly elevated.
•Rough pectoral fin.
• Abdomen is smooth and not
bulging.
•Vent not pinkish.
•Whitish secretion called ‘Milt’
come out on pressing the vent.
Spawner Milter

14. • Selection of female is very important.
• A simple catheter ca be used to find out if the fish is
having eggs or not and shape and size of eggs suggest
that the female is quite ripe.
• Grass carp- red colored eggs
• silver carp- blue
• Color varies with various ecological zones and also
with the age of female.
Healthy, disease free breeders of catla, rohu, mrigal,
silver carp, grass carp and common carp of 2-4 yrs
and 1-5 kgs are normally selected

15. c. Stocking rate (sex ratio)
• Sex composition of breeders is normally maintained as
1:1.
• Males and females are kept separately in brood pond
so that it increases the affinity of mating of the pair
during breeding experiment.
Stocking is done 1500-2000 kg/ha with species
composition of
• 40 surface feeders (Catla 30-20 and silver carp 10-20)+
• 30 column feeders (rohu 20 and grass carp 10)+
• 30 bottom feeders (mrigal 20 and common carp 10).

16. d. Caring the brooders
• Fortnightly or weekly seining- for exercise, general
health checkup and gonadal maturation.
• Frequent replacing breeders from one pond to
another- for food utilization.
• In case of infection- bath in 10 ppm KMno4
e. Manuring and maintenance of water quality in
brood-stocking ponds
•Initial manuring of brood pond- 3 weeks prior to stocking
with cattle dung or compost manure.
•7 days Before fertilization- lime treatment
•1/8th to 1/4th water change frequently.

17. Seining

18. f. Supplementary feeding
• Supplementary feed given daily.
• a complete feed is a proportionate vegetable and animal
protein, carbohydrate and fat. Mixture is prepared from:
half cooked rice and pulse (1:1, 50%)+ oil cake (20%)+ cattle
dung (20%)+ Fish meal/silk worm pupae/ cooked
slaughter house refuse (10%). (Rohu, catla, mrigal,
common carp)
1-2% diet of BW before spawning and 3-4% diet after
spawning. No feed is given for one week before actual
breeding.
Grass carp-fresh aquatic vegetation- Hydrilla, Vallisneria,
Utricularia.
Silver carp- Spirulina, Oscillotoria, Chlorella

19. g. Feeding rate
• Complete stoppage of feed before spawning:
for proper defattening and physical tuning of
breeder.
• Excess feeding may result in heavy
accumulation of mesenteric fat inhibiting the
process of smooth ovulation.

20. Collection of pituitary gland
IMC-serve as Donor
fishes.
However, Common carp
is most preferable
because of its easy
raising.
The glands from fresh
and fully ripe donors are
used.
Suitable period:
breeding season.

21. Preservation and storage of glands
Brazilian Method
Acetone dried
method
Methods

22. Brazilian method of preservation in Abs alcohol
Glands are removed
into absolute alcohol
Transferred to air-
tight phials
After 24 hrs-
Transferred to other air-
tight phials containing
fresh alcohol
Phials are stored in dark air tight
bottle containing ab Al at room
temp in desssicator containing
anhydrous CaCl2 in cool place or
kept inside refrigerator
• Pituitary banks are maintained.
• Since pituitary hormones are water soluble, hence
Brazilian method of preservation in absolute alcohol is
suitable.
Gland remain
effective for 5
years

23. Acetone-dried method
Glands are removed
into acetone and
put into refrigerator
(acetone changed 2-
3 times)
After 36 hrs of
storage, the phials
are taken out and
kept in room
temp.
Glands are removed
and kept on filter paper
to dry
Glands are stored inside air-tight
sterile phials and kept inside
dessicator containg anhydrous
CaCl2 which is then placed inside
a refrigerator
Acetone dried
glands retain
potency for 6
months to 10
years

24. Preparation and preservation of pituitary
extract
• Gland register- serially labeled phials,
details of phial no.
no. and wt of gland and
donor sp’s
Extract: Required quantity of gland → homogenized in
tissue homogenizer →macerated in Physiological
saline (0.3% NaCl)
Intraperitoneal injection: no need of centrifugation
Intramuscular injection: suspension if centrifuged at 1000 rpm for 5
mins

25. Preservation
Glycerine preservation method
1) 1/3 of total volume of extract is made up by adding
DW.
2) Solution is kept in Refrigerator for 24 hrs
3) Glycerine is added to make up 2/3 of volume
(glycerine : water, 2:1)
4) Suspension again stored in refrigerator for 24 hrs
and afterwards centrifuged.
5) Supernatent is filtered and stored in air tight bottles
inside refrigerator
6) Sealed in ampoules (labeled giving details of
hormones conc., date of prep)

26. Dose
Calculation of doses and dilution:
I. 1-3 whole glands from ripe donor to nearly same
wt as that of recipient fish
II. For IMC 2 doses: 2-3 mg/kg BW and 5-8
mg/kg/BW
III. Exotic carps: 3-4 mg/kg/BW, 7-10 mg/kg BW
Weight of spawner Provocative dose
First injection
Final dose
Second injection
0.5- 1 kg 0.5 ml 1 ml
1- 2 kg 0.75 ml 1.5 ml
Above 2 kg 1 ml 2 ml

27. Ponds for induced breeding and the breeding
hapas
• Brood stock
• Brood stock ponds (No blooms, predaceous
insects & fishes.
• In these ponds only breeding hapas are fixed,
inside which the injected brood fishes of both
sexes are released to spawn.
BREEDING HAPA
Box shaped cloth container, like an inverted net with a
cover. (fine-meshed).
Hapa should have fine thin cloth such that ovarian eggs do
not pass out when released by fish

29. Dimensions of hapas
a) 4 kg fish: 3.6X1.8X1.0 m
b) 1.5-4 kg: 2.4X1.2X1.0 m
c) Under 1.5 kg: 1.8X1.0X1.0 m
It has an opening on wider side
It is fixed with the help of bamboo poles
When fixed, bottom of hapa should not touch
muddy pond bottom at least 15-20 cm of hapa
should remain above water level.

30. Injection of pituitary extracts to breeders
• Hypodermic syringe (2ml) with 0.1 ml
graduations is used.
• Thickness and length of needle depends upon
size of recipient fish.
• Generally BDH no. 22 needle- 1-3 kg wt
no. 19 needle – larger fishes
no. 24 needle – smaller fishes
Mode of injection: Intraperitoneal or intramuscular

31. • Intraperitoneal: injected at the base of pelvic
fin. Fish may die due to damage of internal
organ. Practiced in US.
• Tip of needle is inserted under a scale and is
pushed through abdominal wall into body cavity
and then directed parallel to ventral surface

32. Intramuscular: popular in Brazil and India.
Administered in caudal peduncle.
While injecting the needle is first inserted under a scale
parallel to body and muscle is pierced through quickly
at 45°, so that no damage is done to scale

33. • In case of single injection, this could be done
in late in the evening and spawning could be
over by early hours of morning.
• Both injected males and females are kept
together in breeding hapa for spawning.
• In breeding hapa for C.carpio, hanging fibrous
roots of water hyacinth are kept because eggs
of common carp are sticky.

34. Spawning and fertilization
• After few hrs of injection, the brood fishes
spawn naturally in breeding hapa.
• Sex play is observed between male and female
after final injection.
• IMC- spawn within 6-8 hrs
• In case of Chinese carps, stripping is done for
better fertilization

35. Stripping or artificial insemination
• Eggs and milt are forcible extruded out by applying
pressure on the belly of read to breed breeders kept in
hapas.
Methods
Dry method and Wet method
Dry method: After 3-4 hr of second injection, abdomen is
pressed and eggs and milt are collected in tray and
mixed with spoon. After few mins fertilized eggs are
transferred into breeding hapa.
Wet method: physiological saline is used to receive milt
first and eggs later

37. Thank you